Sonic hedgehog-heat shock protein 90β axis promotes the development of nonalcoholic steatohepatitis in mice

Sonic hedgehog (SHH) and heat shock protein 90β (HSP90β) have been implicated in nonalcoholic steatohepatitis (NASH) but their molecular mechanisms of action remain elusive. We find that HSP90β is a key SHH downstream molecule for promoting NASH process. In hepatocytes, SHH reduces HSP90β ubiquitylation through deubiquitylase USP31, thus preventing HSP90β degradation and promoting hepatic lipid synthesis. HSP90β significantly increases in NASH mouse model, leading to secretion of exosomes enriched with miR-28-5p. miR-28-5p directly targetes and decreases Rap1b levels, which in turn promotes NF-κB transcriptional activity in macrophages and stimulates the expression of inflammatory factors. Genetic deletion, pharmacological inhibition of the SHH-HSP90β axis, or delivery of miR-28-5p to macrophages in the male mice liver, impairs NASH symptomatic development. Importantly, there is a markedly higher abundance of miR-28-5p in NASH patient sera. Taken together, the SHH-HSP90β-miR-28-5p axis offers promising therapeutic targets against NASH, and serum miR-28-5p may serve as a NASH diagnostic biomarker.

4. To detect miR-28-5p in human samples, exosomes are extracted from serum at the first.This step may increases the instability of detected data.Can miR-28-5p be extracted and detected directly from serum without affec ng the accuracy of the data?
Minor points: Page 6 lines 162, "absorbed excess lipids", "excess" can be removed.Page 6 lines 168, "caused" should be "causing" Page 7 lines 175, "in mouse blood", do the authors men "in the serum"?Page 7 lines 179 and Page 8 lines 215, "Compared to" should be "Compared with" Page 7 lines 201, "I Importantly" should be "Importantly" Page 13 lines 355, Abbrevia ons appearing for the first me (NPs) should be given in full text (nanopar cles) Page 13 lines 356, "The par cle size of NPs was characterized were approximately 100 nm" needs to be revised.

Comments to Authors
This manuscript inves gates the role of HSP90β in Sonic hedgehog (SHH) mediated NASH development.The authors extend their previous studies on inves ga ng SHH signaling mechanisms to iden fy downstream mediators.Here the inves gators show that HSP90β is cri cal in SHH mediated fa y liver development and inflamma on.The authors u lize in vivo and in vitro strategies to unravel the role of SHH-HSP90β-miR-28-5p axis in NASH, however several concerns listed below, in methodologies and lack of clarity in results presented, dampen enthusiasm.Overall the data presented lack robust outcomes to jus fy hepatocyte specific SHH-HSP90β signaling in NASH.
1) Experiments in Figure 1 u lize cyclopamine, pharmacological inhibitor of SMO, to determine the effect of SHH signaling on murine NASH development.Using this inhibitor, inves gators also evaluate altera ons in HSP90β in Figure 2. Addi onal specific inhibi on strategies of SHH signaling such as siRNA must be used for robust analysis, since iden fica on of SHH-specific HSP90β changes is based on this inhibi on.
2) In Figure 2, in vivo and in vitro approaches show that HSP90β protein is increased by SHH in HFFClivers and primary hepatocytes as well as HEPA 1-6 cells and HEPG2 cells.However, it is not clear if this increase in HSP90β occurs in hepatocytes only.Whether HSP90β is altered in liver macrophages and stellate cells must be evaluated.It is important to demonstrate cell-type specificity in HSP90β to jus fy subsequent genera on of hepatocyte-specific HSP90β KO mice.
3) Figure 2 I-N shows HSP90β and HSP90α levels in human liver samples.It is not clear if the liver samples are NAFLD or NASH pa ents?Also lack of representa ve IHC micrographs make it difficult to discern the levels shown in the graphs.4) HSP90β flox/flox mice were generated commercially.Data must be shown to confirm specificity of these mice.Further to genotype hepatocyte-specific HSP90β KO mice Fig S2B confirms that HSP90β is not expressed in liver ssues of KO mice.This is intriguing since HSP90β is a cons tu ve form of HSP90 and expressed in several cell types in the liver including macrophages.To confirm hepatocyte specific KO, HSP90β must be decreased or lost in hepatocytes whereas other liver cells should exhibit HSP90β expression.5) Figure S5 u lizes 17-AAG in vivo as a HSP90 inhibitor to confirm the specificity of HSP90β in murine NASH.17-AAG can inhibit HSP90α and HSP90β in various cell types.Addi onal strategies to target HSP90β using siRNA must be employed to determine SHH signaling in hepatocytes.6) Overexpression of SHH using ADV-SHH was demonstrated by detec on of GFP+ signals in livers (Fir S6A).It is not clear which cells overexpress SHH and the magnitude of overexpression?7) Much of the work on HSP90β deubiquityla on is performed in hepatocyte cell lines.These studies must be confirmed in primary hepatocytes and changes in HSP90β deubiquityla on must be demonstrated in NASH hepatocytes.EMSA analysis using BMAL1 an bodies (Fig 5D) is not convincing.8) SHH induced inflamma on in NASH is a ributed to HSP90β facilitated extracellular communica on by liver exosomes.The protocol used to prepare liver exosomes in not clear.This group has previously published prepara on of exosomes from adipose ssue and the same method is extended to the liver.It is not clear if these purified liver exosomes capture the disease related phenotype.
Reviewer #3 (Remarks to the Author): Your elegant studies reveal a novel Hedgehog signaling mechanism that has poten ally broad implica ons, including induc on of a pro-inflammatory macrophage phenotype that contributes to the pathogenesis of nonalcoholic steatohepa s (NASH) and related liver fibrosis.
Briefly, your work complements and extends earlier work by other groups showing that both hepa c expression of SHH ligand and SHH-ini ated down-stream signaling that ac vates Smoothened (Smo) are increased in people and mice with NASH.Importantly, your studies show that systemic administra on of cyclopamine (Cp), a direct inhibitor of Smo, improves diet-induced steatosis, inflamma on and fibrosis in mouse models.Although not cited in your manuscript, the inhibitory effects of direct Smo antagonism on murine liver inflamma on and fibrosis had been reported previously and a ributed to paracrine effects of hepatocyte-derived HH ligands on other types of HH-responsive liver cells (PMID: 21912653).However, the earlier study did not inves gate the possible role of autocrine hepatocyte HH signaling in NASH pathogenesis.
Your work demonstrates that SHH-ini ated ac va on of Smo in hepatocytes permits Bmal1 (a circadian clock-associated transcrip on factor) to disassociate from SuFu.Your result complements and extends data from another group which showed that hepatocyte Smo ac vity regulates the circadian clock (PMCID PMC6146234).You have significantly advanced understanding of the mechanisms involved by demonstra ng that following Smo ac va on and release from SuFu,"free" Bmal1 localizes to nuclei where it promotes the transcrip on of a deubiqui nase (Usp 31) that stabilizes the heatshock chaperone, Hsp90b.Hsp90b, in turn, facilitates the release of hepatocyte-derived exosomes carrying miR 28-5p and this mIR suppresses Rap1b in macrophages, resul ng in ac va on of NF-KB signaling and increased macrophage produc on of several pro-inflammatory cytokines that are known to promote insulin resistance, hepa c steatosis, lipotoxicity and fibrogenic ac va on of hepa c stellate cells.Based on these results and evidence that SHH and mir28-5p are increased in humans with NASH, you suggest that miR28-5p might be both a novel biomarker and therapeu c target in NASH.The data are compelling and as you state, have ac onable implica ons clinically.However, a few issues require clarifica on: 1 -Fig 2I is confusing as presented.More granular informa on is necessary to assess correla ons between expression of SHH and Hsp90B and NASH severity.For example, how do levels of SHH/Hsp90b correlate with the severity of the NAFLD Ac vity Score (NAS) and fibrosis stage?The NAS is typically assessed to judging the severity of hepa c steatosis (graded 0-3), liver inflamma on (graded 0-2) and hepatocyte ballooning (graded 0-2).Fibrosis staging (generally scored 0-4) is used to assess fibrosis severity.This informa on is par cularly important for your studies because SHH is known to accumulate in ballooned hepatocytes and the severity of hepatocyte ballooning and liver fibrosis severity are significantly correlated.
2 -You suggest that circula ng miR28-5p might be a novel biomarker for NASH.You also show that serum levels of SHH increase in NASH and that SHH ini ates the process that leads to increased miR28-5p.Is miR28-55p superior to SHH for "predic ng" severity of NASH or fibrosis?3 -Some of the approaches that you used to manipulate HH signaling (e.g., systemic administra on of Cp or Adenoviral vector delivery of SHH ligand) are not hepatocyte-specific and so, would impact pathway ac vity in mul ple cell types both in liver and other ssues.While your in vitro experiments clearly demonstrate that SHH can induce Hsp90b in hepatocytes and your in vivo experiments prove that hepatocyte derived Hsp90b cri cally mediates liver inflamma on and fibrosis, can you assure that SHH is the main mediator of Hsp90b accumula on in hepatocytes in your mouse NASH models (or human NASH liver samples)?What was the ra onale for using ADV-SHH in mice with diet-induced NASH given that your data and work of others shows that endogenous SHH expression is increased in NASH? 4 -Previous publica ons have shown that liver cell-derived exosomes carry SHH and Indian Hedgehog (PMCID PMC3724240).Studies have also reported that hepatocyte-derived IHH is important for ac va ng macrophages and promo ng hepa c stellate cell ac va on in mouse models of NASH (PMCID PMC5226184).Do the exosomes that carry miR25p also carry either of the HH ligands?If so, is the HH ligand cargo required to miR28-5p to promote the proinflammatory macrophage phenotype?hepa c lipid accumula on with recent work from at least two other groups demonstra ng that targeted inhibi on of hepatocyte Smo promotes hepatocyte lipid accumula on (PMCID PMC4869931, PMID PMC8559255)? 5 -Others have reported that Smo ac vity inhibits adipogenesis but you found that systemic exposure to Cp (a Smo antagonist) reduced adiposity in mice with diet-induced obesity and NASH.How might this paradox be explained?
We are grateful for the reviewers' constructive comments and suggestions and particularly appreciated that a number strengths were identified in our work.Here, we have attempted to address all the reviewers' concerns from the original submission point-to point.The reviewers' comments are provided in bold italics below with our response following.

Reviewer #1:
This is a very interesting and novel study revealing that SHH promotes NASH process though HSP90/3.To demonstrate this point, the authors used different mouse models, as well as patient samples to find out specific role of HSP90/3 in NASH development.The authors provided extensive set of data showing that USP31, which is transcriptionally regulated by Shh-Bmal1, was a novel DUB of HSP90/3.The authors also found HSP90/3 increased secretion of exosomes enriched with miR-28-5p, which promoted inflammatory response in macrophages.Clinical data also showed serum miR-28-5p correlated quite well with NASH.Finally, the authors used nanoparticles wrapping miR-28-5p antagomir decrease inflammation in HFFC diet-induced NASH mouse.Overall, I find the study to be compelling and well suited for Nature Communications.However, there are a few points that should be addressed prior to publication.Major points: We appreciate the reviewer's deeply insightful evaluation.We explained the all the questions reviewer concerned and some of them were added in discussion of our manuscript.
1. Since activation of the Shh pathway promoted Bmal1's nuclear translocation, is there any relevance between circadian rhythm and NASH?Please discuss this.
Circadian misalignment has been identified as a risk factor for metabolic disease (Reinke and Asher, 2016).The circadian clock is involved in regulation of hepatic triglyceride accumulation, inflammation, oxidative stress, and mitochondrial dysfunction (Adamovich et al., 2014;Hatori et al., 2012;Jacobi et al., 2015).Bmal1, as a core circadian clock gene, play an important role in NAFLD.Bmal1 deficiency protects against steatohepatitis, inflammation, and fibrosis, preventing the development of NASH (Jouffe et al., 2022).We added some discussion in the revised manuscript.
2. Homemade nanoparticles in this manuscript is characterized with good miRNA-packing capacity and particle size, mainly accumulating in the liver.The authors need to briefly discuss the ability of NPs to enter macrophages in the liver.
Some studies suggested that nanoparticles can undergo transcytosis through cells in the liver, especially in Kupffer cells (Soji et al., 1992;Ogawara et al., 1999).The method of nanoparticle production in this manuscript has been described in our previous work.Our previous data demonstrated that the nanoparticle boosted the accumulation of its cargo in the liver of the HFD-fed obese mice, particularly in the activated macrophages (Shen et al., 2020).
3. Is there any researches about whether miR-28-5p has effects on hepatocytes or hepatic stellate cells.If there any, do these known miR-28-5p targeted genes participate in NASH process?
It was reported that miR-28-5p was downregulated in tumors (Lv et al., 2019) and cancer stem cells (Xia et al., 2019).IGF1 has been found as the direct target of miR-28-5p (Xia et al., 2019;Shi and Teng, 2015).The level of IGF-1 is down-regulated in NAFLD patients compared to healthy controls (Yao et al., 2019), this is in consistent with our finding that miR-28-5p was upregulated in NAFLD/NASH patients (Fig. 6h).In addition, miR-28-5p upregulated the expression of ATP-binding cassette transporter A1 (ABCA1) via the inhibition of ERK2 in HepG2 cells (Liu et al., 2016).The plasma levels of miR-28-5p were significantly increased in unstable angina patients (Liu et al., 2015).However, the correlation between miR-28-5p and NASH has not been reported.4. To detect miR-28-5p in human samples, exosomes are extracted from serum at the first.This step may increases the instability of detected data.Can miR-28-5p be extracted and detected directly from serum without affecting the accuracy of the data?
In this manuscript, we found that miR-28-5p in hepatocytes-derived exosomes promoted inflammatory gene expression in macrophages.HSP90 is critical for the maturation and secretion of exosomes, while no studies have shown that HSP90 affects the secretion of other extracellular vesicles.
To avoid the contamination of miRNAs from other extracellular vesicles, we extracted miRNAs directly from purified exosomes.

Minor points:
Page 6 lines 162, "absorbed excess lipids", "excess" can be removed.Page 6 lines 168, "caused" should be "causing" Page 7 lines 175, "in mouse blood", do the authors men "in the serum"?Page 7 lines 179 and Page 8 lines 215, "Compared to" should be "Compared with" Page 7 lines 201, "I Importantly" should be "Importantly" Page 13 lines 355, Abbreviations appearing for the first time (NPs) should be given in full text (nanoparticles) Page 13 lines 356, "The particle size of NPs was characterized were approximately 100 nm" needs to be revised.
We are very grateful to the reviewer for reviewing the manuscript carefully and discovering some of the mistakes.We revised the text accordingly in the new manuscript.

Reviewer #2:
This manuscript investigates the role of HSP90/3 in Sonic hedgehog We are very grateful to the reviewer for reviewing the manuscript carefully.We performed the experiments as suggested to address the reviewer's concerns.We performed siRNA experiments as the reviewer suggested, the results were added in the revised manuscript.Similar as Smo inhibitor, knocking down Smo resulted in decreased Hsp90β expression by promoting its ubiquitylation (revised manuscript, Fig. 2j-k, and 4d).

1) Experiments in
2) In Figure 2, in vivo and in vitro approaches show that HSP90β protein is increased by SHH in HFFC-livers and primary hepatocytes as well as HEPA 1-6 cells and HEPG2 cells.However, it is not clear if this increase in HSP90/3 occurs in hepatocytes only.Whether HSP90/3 is altered in liver macrophages and stellate cells must be evaluated.It is important to demonstrate cell-type specificity in HSP90/3 to justify subsequent generation of hepatocyte-specific HSP90/3 KO mice.
We checked the expression of Hsp90j3 in the isolated hepatocytes, Kupffer cells and hepatic stellate cellsfrom mice liver.Then we checked the Hsp90 expression in the presence of Shh or compared between normal chow diet and HFFC diet treatment.Hsp90j3 were only upregulated in hepatocytes from HFFC-fed mice liver.Similarly, Hsp90j3 expression were only upregulated by Shh treatment in hepatocytes but not changed in Kupffer cells and hepatic stellate cells (revised manuscript, Fig. 2c and d).Taken together, these data suggested that Hsp90j3 overexpression was hepatocyte-specific in a NASH mouse model.Thus, we generated hepatocyte-specific Hsp90j3 KO mice to evaluate its role in NASH development.
3) Figure 2 I-N shows HSP90/3 and HSP90a levels in human liver samples.It is not clear if the liver samples are NAFLD or NASH patients?Also lack of representative IHC micrographs make it difficult to discern the levels shown in the graphs.
We thank the reviewer to point this out.We re-organized all the clinical data, and categorized into three different groups: healthy controls, NAFLD and NASH patients.When compared among these groups, the expression levels of HSP90j3 were positively correlated with disease progression, while the expression of HSP90a was not related to disease progression (revised manuscript, Fig. 2m-o).is not expressed in liver tissues of KO mice.This is intriguing since HSP90/3 is a constitutive form of HSP90 and expressed in several cell types in the liver including macrophages.To confirm hepatocyte specific KO, HSP90/3 must be decreased or lost in hepatocytes whereas other liver cells should exhibit HSP90/3 expression.
The western blot bands were developed with short exposure time.When exposed for a longer period, we could see Hsp90j3 bands in livers of
We further verified the expression levels of Hsp90j3 protein in hepatocytes, Kupffer cells and hepatic stellate cells, isolated from Hsp90I3 3Hep mice.The results showed that only hepatocyte Hsp90j3 was eliminated (revised manuscript, Fig. S2c).
Response Figure 1 Western blot analysis of Hsp90j3 in the liver tissues of male or female Hsp90j3 fl/fl and Hsp90I3 3Hep mice.
5) Figure S5 utilizes 17-AAG in vivo as a HSP90 inhibitor to confirm the specificity of HSP90/3 in murine NASH.17-AAG can inhibit HSP90a and HSP90/3 in various cell types.Additional strategies to target HSP90/3 using siRNA must be employed to determine SHH signaling in hepatocytes.
Figure S5 demonstrates that low-dose 17AAG improved the NASH phenotype, similar to that in Hsp90I3 3Hep mice.In this manuscript, we found that Hsp90j3was mediated by SHH pathway.However, as reviewer mentioned, whether SHH signaling is affected by Hsp90j3is unknown.Instead of using siRNA, we isolated primary hepatocytes from Hsp90I3 3Hep mice.It turned out that there was no significance of Gli1 gene expression in primary hepatocytes (Response Figure 2).Taken all our data together, we concluded that Hsp90β was the downstream of SHH signaling pathway.
Response Figure 2 Gli1 gene expression in primary hepatocytes of ADV virus is easy to infect hepatocytes and Kupffer cells, while it is hard to infect immunity cells (B cells, T cells, etc) and HSCs.As Liu Qiongming investigated, sh-control or Pu.1 adenovirus transduces both hepatocytes and Kupffer cells without affecting Pu.1 expression in B cells, T cells, or HSCs (Liu et al., 2020).Thus, in order to confirm the infection and expression of ADV in the liver, we evaluate their infection efficiency by flow cytometry.We found that in all the GFP+ cells, around 84.8% expressed albumin, a hepatocyte marker, while 7.26% GFP+ cells expressed F4/80, a macrophage marker.These results suggested that most of the cells that overexpressed Shh are hepatocytes.
Response Figure 3 Mice were administered ADV-Shh-GFP.Liver cells were analyzed by flow cytometry to evaluate their infection efficiency.
7) Much of the work on HSP90/3 deubiquitylation is performed in hepatocyte cell lines.These studies must be confirmed in primary hepatocytes and changes in HSP90/3 deubiquitylation must be demonstrated in NASH hepatocytes.EMSA analysis using BMAL1 antibodies (Fig 5D) is not convincing.
As the reviewer suggested, we performed the Hsp90j3 deubiquitylation experiments in primary hepatocytes.As expected, Usp31 knockdown reversed the reduction of Hsp90j3 ubiquitylation (revised manuscript, Fig. 4i   and j).In vitro ubiquitination experiments showed that Hsp90j3 ubiquitylation decreased when incubated with the primary hepatocyte lysates treated with Shh or isolated from HFFC-diet mice (revised manuscript, Fig. 4e and S7f).
The Hsp90j3 ubiquitylation in primary hepatocytes isolated from HFFC-diet mice also decreased (revised manuscript, Fig. 4c).EMSA analysis was reperformed in primary hepatocytes and replaced the previous data (revised manuscript, Fig. 5d).

8) SHH induced inflammation in NASH is attributed to HSP90β
facilitated extracellular communication by liver exosomes.The protocol used to prepare liver exosomes in not clear.This group has previously published preparation of exosomes from adipose tissue and the same method is extended to the liver.It is not clear if these purified liver exosomes capture the disease related phenotype.
A reference (Matejovič et al., 2021) to the protocol for extracting exosomes from the liver described in our method was not included.In this reference, three protocols of EV extraction from livers were compared.It turned out that the Protocol C, which was used in this manuscript, seemed to contain a higher amount of smaller size EVs (mean vesicle size: 117 ± 40.6 nm) with an EV-like morphology.We also referred to this article, as Protocol C perfectly met our needs, and the method was consistent with that from adipose tissues we quoted.We thank the reviewer to point this out.We re-organized all the clinical data, and categorized into three different groups: healthy controls, NAFLD and NASH patients.When compared among these groups, the expression levels of HSP90β were positively correlated with disease progression, while the expression of HSP90α was not related to disease progression (revised manuscript, Fig. 2m-o).

Reference
2 -You suggest that circulating miR28-5p might be a novel biomarker for NASH.You also show that serum levels of SHH increase in NASH and that SHH initiates the process that leads to increased miR28-5p.Is miR28-5p superior to SHH for "predicting" severity of NASH or fibrosis?
When extracting exosomes from clinical human serum samples, we also tested the concentration of SHH in the serum with an ELISA kit.Although the average concentration of SHH in the serum of NASH patients was higher than healthy controls, but there was no significant difference between the two groups (revised manuscript, Fig. 6i).In normal group, SHH concentration of 4 volunteers was far higher than others in the same group.As reported, SHH secretion elevated not only in NASH but also in pulmonary fibrosis (Kugler et al., 2015), chronic kidney disease (Zhou et al., 2016), and alcohol-associated liver injury (Kumar et al., 2023).Therefore, we think that miR-28-5p in exosomes from serum serves as a better marker to predict the severity of NASH patients.In vitro, Shh ligand promotes the expression of Hsp9013, in primary hepatocytes, Hepa 1-6 and AML12 cells (revised manuscript, Fig. 2d-i).In vivo, Shh overexpression in liver promoted the Hsp9013 protein levels (revised manuscript, Fig. 3b).Before we focused on Shh, we checked a series of NASH risk factors, such as PA and OA, cytokines IL-113, cytokines TNF-a, LPS, hydrogen peroxide, glucose and insulin, in regulating Hsp9013 protein levels in Hepa 1-6 cells, none of the treatments promoted Hsp9013 protein levels (revised manuscript, Fig. S7a and b).Thus, we believed that Shh played an very important role in mediating Hsp9013 expression.Shh exists in exosomes derived from livers of normal or NASH mice.In addition, the Shh ligand is also known to stimulate hepatic stellate cells and fibroblasts via a paracrine mechanism, thereby promoting profibrogenic response in mouse model of NASH (Kwon et al., 2016;Rangwala et al., 2011;Jung et al., 2010;Choi et al., 2009;Hirsova et al., 2013;Omenetti et al., 2008).
However, this article only proves Ihh activated HSC, but did not mention whether Ihh activated macrophages.
To study the effects of Shh on macrophages, BMDM cells were treated with Shh, no proinflammatory phenotypes were observed (revised manuscript, Fig. 6a).In contrast, when BMDM cells were treated with miR-28-5p mimics alone, the gene expression of pro-inflammatory cytokines was increased (revised manuscript, Fig. 6e).Taken together, we believe that miR-28-5p, but not Shh, promoted the proinflammatory macrophage phenotypes.
4 -Can you rectify your findings which suggest that stimulating Smo activity in hepatocytes promotes hepatic lipid accumulation with recent work from at least two other groups demonstrating that targeted inhibition of hepatocyte Smo promotes hepatocyte lipid accumulation (PMCID PMC4869931, PMID PMC8559255)?
Of the two recent works reviewer mentioned, one of the articles (PMID PMC8559255) could not be found, probably because of the wrong PMC number.In another work (Matz-Soja et al., 2016), hepatocyte-specific Smo deletion mice of 8 weeks old were fed with normal diet.In adult healthy liver, SHH signaling is inactive (Hirose et al., 2009;Machado and Diehl, 2018), thus, the effect of Smo KO would not significantly affect Shh signaling pathway further.Shh is reactivated when the liver is injured, or HFFC-diet feeding as described in our work.This article mentioned that "The potential of impaired Hh signaling to trigger steatosis independent of nutritional changes suggests that malfunctions in this pathway may pave the way for the development of NAFLD long before other cues may lead to further aggravation."This statement might not reflect the progression of NASH, since Shh expression is higher in livers from both NASH patients and NASH animal models.
In our manuscript, we found that Shh increased Hsp90j3 protein levels in hepatocytes, while studies have showed that Hsp90j3 promotes de novo lipid synthesis in hepatocytes by increasing the expression of SREBP (Zheng et al., 2019;Kuan et al., 2017).Therefore, we think lipid accumulation reduction in our report is mainly due to the reduced SREBPs and de novo lipogenesis in the liver.
5 -Others have reported that Smo activity inhibits adipogenesis but you found that systemic exposure to Cp (a Smo antagonist) reduced adiposity in mice with diet-induced obesity and NASH.How might this paradox be explained?
As the reviewer did not provide the PMCID of the research, we find an article closed to this (Suh et al., 2006).In vitro, SHH treatment in 3T3-L1 cells prevented adipogenesis.At the same time, the authors found that in HFD-induced obese mice, the gene expression of Smo, Gli1, Gli2, and Gli3 were significantly decreased, indicating that SHH pathway was inhibited.In the absence of Shh, Smo activity is repressed by PTCH1 (Teperino et al., 2014), therefore under these conditions, administration of Cp (a Smo antagonist) might not cause significant effects on SHH pathway.
We further checked the distribution of Cp in vivo.Cp was injected intraperitoneally, after 1 hour, Cp concentration was detected with HPLCmass spectrometry.Using semi-quantitative detection of peak areas, it was found that the concentration of Cp in adipose tissues was only 24.4% of that in the liver (Response Figure 4).These data suggested that Cp mainly function in the liver.
In this manuscript, we also found that Cp prevented lipid accumulation by decreased de novo lipid synthesis in liver and we believed that the weight loss of mice after Cp treatment was mainly because of that.
Response Figure 4 Mice were injected intraperitoneally with Cp and its concentration was detected with HPLC-mass spectrometry.

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author): In this revised manuscript, the authors have done plenty of experiments including detec on of miR-28-5p in human samples .They have successfully addressed my concerns and I have no further ques ons.
Reviewer #3 (Remarks to the Author): Your work iden fies a novel mechanism whereby Sonic hedgehog ligand (SHH) ac vates Smoothened and Gli1-dependent canonical hedgehog signaling in liver cells to promote the pathogenesis of NASH.
Reviewers of the ini al version of the manuscript had some ques ons and concerns that you have largely addressed via addi onal experiments.This informa on has been incorporated into the revised manuscript and provides addi onal support for your model.I have not further concerns.
Reviewer #4 (Remarks to the Author): In this study, Zhang et al. suggest that the ac va on of the Sonic hedgehog pathway promotes Hsp90β stability and causes NASH.
Many results, however, argue against the authors' hypothesis.
For instance, the authors state "We further checked Hsp90β expression isolated primary hepatocytes (HPs), Kupffer cells (KCs) and hepa c cells (HSCs) from mice fed with chow or HFFC diet for 16 weeks.It turned out that Hsp90β only increased in primary hepatocytes (Fig. 2c).Likewise, Shh only promoted Hsp90β protein levels in HPs".
Indeed, by defini on NASH is a histological diagnosis made when steatosis ≥5%, hepatocyte ballooning and inflamma on are simultaneously present.Moreover, liver fibrosis staging 1 to 3 is a concomitant feature.Instead, Hsp90β was found to increase only in primary hepatocytes sugges ng no role of this heat shock protein in inflamma on and in fibrosis.
The authors observe "We found that HSP90β was posi vely correlated with disease progression, while the expression of HSP90a was not related to disease progression (Fig. 2m-o)".Indeed, in Figure 2 panel n the HSP90β levels were similar in NAFL and NASH, therefore I could not appreciate a "posi ve correla on with disease progression".
The models used in this study do not replicate what happens in the liver during NASH development.
Instead of doing hepatocyte Hsp90β abla on to observe effects on steatohepa s, a good study design would have included organoids where ssue architecture is maintained and interac ons/crosstalk between different cell types is present.
What does "Liver ssues from Hsp90β fl/fl or Hsp90βΔHep 648 mice were obtained" mean?Reading carefully the manuscript, I realized that the authors worked only on isolated cells where the gene codifying for HSP90β was suppressed or overexpressed.Thus, they did not use condi onal KO or transgenic mice.

"Gene knockdown and overexpression
For transient transfec cells were transfected with indicated miRNA or siRNA constructs (Genepharma, Shanghai, China) using RNAiMAX (Invitrogen, California, USA), or indicated plasmids using the Lipofectamine 3000 (Invitrogen, California, USA) reagent following the manufacturer's instruc ons." Overall, this study does not show causality but just correla ons.
In addi on, it is clear that the manuscript was prepared for another journal where methods precede the results.In fact, the results sec on contains a series of abbrevia ons that are reported only in the methods.One for all, "high fat, high fructose, and high cholesterol diet (HFFC)".
Finally, please do not address people as "obese", like in lines 76 and 77 "pa ents and obese mice" and "overweight and obese children", but rather say children with overweight or obesity, this to avoid obesity s gma.
We acknowledge all the reviewers' efforts to improve our manuscript.As the review 1 and 3 are satisfactory with our response and revised manuscript, while the reviewer 2did not respond during the first revision round, here, we have attempted to address the reviewer 4' concerns point-to point.The reviewer 4' comments are provided in bold italics below with our response following.
Reviewer #4 (Remarks to the Author): In this study, Zhang et al. suggest that the activation of the Sonic hedgehog pathway promotes Hsp90/3 stability and causes NASH.
Many results, however, argue against the authors' hypothesis.
We appreciate the reviewer's deeply insightful comments.We explained all the questions reviewer concerned and demonstrate the existence of intercellular communication through organoid experiments as reviewer suggested.
Indeed, by definition NASH is a histological diagnosis made when steatosis 5%, hepatocyte ballooning and inflammation are simultaneously present.Moreover, liver fibrosis staging 1 to 3 is a concomitant feature.Instead, Hsp90/3 was found to increase only in primary hepatocytes suggesting no role of this heat shock protein in inflammation and in fibrosis.
In this manuscript, we discovered that HSP90β only elevated in hepatocytes and played an indirect role in promoting inflammation and fibrosis.In murine and human liver cell lines and primary hepatocytes, Hsp90P was increased after Shh treatment (Fig. 2g-i).The expression levels of HSP90P were also increased in NASH patients, compared with healthy controls (Fig. 2n-o).In vivo, hepatocyte-specific Hsp90P ablation reversed inflammation and fibrosis in the liver induced by HFFC (Supplementary Fig. 4a-e).To understand the mechanism how hepatic HSP90P affected the inflammation in macrophages, we analyzed the cell-cell communications in the animal models.It turned out that the increased HSP90P protein in hepatocytes promoted the secretion of exosomes enriched with miR-28-5p, which stimulated NF-KB transcriptional activity in macrophages and increased the expression of inflammatory factors (Fig. 6c-e).In vivo, targeted delivery of miR-28-5p antagomir to hepatic macrophages mitigated the HFFC-induced hepatic damage and inflammation, which suppressed the development of NASH (Fig. 8b-k).Taken together, as the reviewer suggested, hepatic Hsp90P has no direct effects on inflammation and fibrosis, but rather through exosomes enriched with miR-28-5p.
2.The authors observe "We found that HSP90/3 was positively correlated with disease progression, while the expression of HSP90a was not related to disease progression (Fig. 2m-o)".Indeed, in Figure 2 panel n the HSP90/3 levels were similar in NAFL and NASH, therefore I could not appreciate a "positive correlation with disease progression".
We thank reviewer for pointing this out.In this manuscript, we generated tissue-specific KO mice (Hsp90β ΔHe p) by crossing Hsp90β fl/fl with Alb-Cre mice (Supplementary Fig. 2).Hsp90β levels in various cell types from livers were examined after genotyping (Supplementary Fig. 2b and c).The genotype of 18 mice for the experiments was confirmed (Supplementary Fig. 2d).Hsp90βfl/fl and Hsp90β ΔHep mice were used to discover the role of Hsp90β in NASH development (Fig. 3a-h and Supplementary Fig. 3a-k, 4a-e and 6a-g).The paragraph that the reviewer mentioned is to describe how we isolate exsosomes from mice hepatocytes with different genetic backround.The aim of this study is to investigate the cell-cell communications via secreted exsosomes.Hepatic Hsp90β per se did not affect inflammation response, however, Hsp90β facilitate the secretion of exsosomes from the liver that were enriched with miR-28-5p, miR-28-5p then activated the inflammatory response in macrophages (Fig. 6b-f).

5."Gene knockdown and overexpression
For transient transfection, cells were transfected with indicated miRNA or siRNA constructs (Genepharma, Shanghai, China) using RNAiMAX (Invitrogen, California, USA), or indicated plasmids using the Lipofectamine 3000 (Invitrogen, California, USA) reagent following the manufacturer's instructions."Overall, this study does not show causality but just correlations.
This part of the method has been corrected to "To demonstrate the impacts of shh pathway on HSP90β protein levels, primary hepatocytes, Hepa 1-6 or HepG2 cells were transfected with indicated siRNA (Genepharma, Shanghai, China) using RNAiMAX (Invitrogen, California, USA) following the manufacturer's instructions.To perform co-immunoprecipitation or in vitro deubiquitination assay, indicated plasmids were transfected with Lipofectamine 3000 in primary hepatocytes, Hepa 1-6 or 293T cells following the manufacturer's instructions.To study the role of miR-28-5p in regulating inflammation in macrophages, BMDM cells were transfected with miR-28-5p mimic or miR-28-5p antagomir using RNAiMAX (Invitrogen, California, USA) following the manufacturer's instructions." 6.In addition, it is clear that the manuscript was prepared for another journal where methods precede the results.In fact, the results section contains a series of abbreviations that are reported only in the methods.One for all, "high fat, high fructose, and high cholesterol diet (HFFC)".Finally, please do not address people as "obese", like in lines 76 and 77 "patients and obese mice" and "overweight and obese children", but rather say children with overweight or obesity, this to avoid obesity stigma.
We thank reviewer for pointing these out.We have corrected these issues as the reviewer suggested.

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SHH) mediated NASH development.The authors extend their previous studies on investigating SHH signaling mechanisms to identify downstream mediators.Here the investigators show that HSP90/3 is critical in SHH mediated fatty liver development and inflammation.The authors utilize in vivo and in vitro strategies to unravel the role of SHH-HSP90/3-miR-28-5p axis in NASH, however several concerns listed below, in methodologies and lack of clarity in results presented, dampen enthusiasm.Overall the data presented lack robust outcomes to justify hepatocyte specific SHH-HSP90/3 signaling in NASH.
Figure 1 utilize cyclopamine, pharmacological inhibitor of SMO, to determine the effect of SHH signaling on murine NASH development.Using this inhibitor, investigators also evaluate alterations in HSP90/3 in Figure 2. Additional specific inhibition strategies of SHH signaling such as siRNA must be used for robust analysis, since identification of SHH-specific HSP90/3 changes is based on this inhibition.
4) HSP90/3 flox/flox mice were generated commercially.Data must be shown to confirm specificity of these mice.Further to genotype hepatocyte-specific HSP90/3 KO mice Fig S2B confirms that HSP90/3 Hsp90βfl/fl and Hsp90β ΔHe p mice.6) Overexpression of SHH using ADV-SHH was demonstrated by detectionof GFP+ signals in livers (Fir S6A).It is not clear which cells overexpress SHH and the magnitude of overexpression?
Extracellular vesicles (EVs) act as a signaling mediator, resulting in lipid accumulation, macrophage and hepatic stellate cell activation, further promoting inflammation and liver fibrosis progression during the development of NAFL/NASH (Xu X, Poulsen KL, Wu L, Liu S, Miyata T, Song Q, Wei Q, Zhao C, Lin C, Yang J. Targeted therapeutics and novel signaling pathways in non-alcohol-associated fatty liver/steatohepatitis (NAFL/NASH).Signal Transduct Target Ther.2022 Aug 13;7(1):287.).In this manuscript, we discovered that exosomes enriched with miR-28-5p secreted by hepatocytes promoted the expression of inflammatory factors in macrophages.The crosstalk between hepatocytes and macrophages was proved by extracting liver exosomes from Hsp90βfl/fl or Hsp90βΔHep mice with chow or HFFC diet, and then detecting the inflammatory factors in BMDM cells after treated with hepatocytesderived exosomes (Fig.6c).We also performed liver organoids experiments as the reviewer suggested.It turned out that the results were in consistent with cell co-culture experiments.Shh promoted inflammatory gene expression in organoid derived from Hsp90β fl/fl mice.Increased Il-1β, Tnf-α and Il-6 expression could be blunted either in hepatic organoid derived from liver-specific Hsp90 knockout mice (Hsp90β ΔHe p), or in hepatic organoid derived from Hsp90β fl/fl mice treated with miR-28-5p antagomir (Supplementary Figure11).

4.
What does "Liver tissues from Hsp90/3 fl/fl or Hsp90/3ΔHep 648 mice were obtained" mean?Reading carefully the manuscript, I realized that the authors worked only on isolated cells where the gene codifying for HSP90/3 was suppressed or overexpressed.Thus, they did not use conditional KO or transgenic mice.